PROJECT SUMMARY Rotavirus (RV) remains the leading cause of death due to diarrheal disease in children worldwide, with a disproportionate burden of severe and fatal disease in low-income countries of sub-Saharan Africa and Asia. Oral RV vaccines such as Rotarix (GlaxoSmithKline) are highly efficacious in high-income countries, but for unclear reasons they significantly underperform in low-income countries, a significant obstacle to the reduction of diarrheal disease worldwide. Greater understanding of why oral vaccines fail in these settings is needed to improve vaccine performance and develop next-generation vaccines. The goal of this study is to refine approaches for assessing immunological responses to oral vaccines, using rotavirus (RV) as a prototype. Generation of pathogen-specific immunological memory is the fundamental goal of vaccination, but little is known about the ability of RV vaccines to do this. Since antibody responses have consistently been shown to be critical for RV immunity, assessment of B cell responses to RV vaccination is paramount, particularly the generation of RV-specific memory B cells. However, evaluations of RV-specific lymphocyte responses to currently licensed vaccines are completely lacking. The hypothesis is that under proper conditions (e.g. in high- income countries), circulating RV-specific B cell subsets reflecting immune memory can be identified following vaccination. Similarly, the project proposes that oral RV vaccine underperformance in low-income settings is due to a failure of the vaccine to generate memory B cell responses, and that this may be mediated in part by early B cell exhaustion due to the increased gut pathogen burden unique to these settings. To test these hypotheses, flow cytometry will be used to define the immunophenotype of circulating RV- specific B among infants in Burlington, VT, USA and Dhaka, Bangladesh following oral RV vaccination. These cohorts represent populations in which vaccine responses are excellent (VT) and diminished (Bangladesh). RV-specific subsets thus identified will be correlated with serum antibody responses and fecal vaccine shedding, surrogate markers of vaccine effect. Next, the project will evaluate the contribution of cofactors thought to impact RV vaccine performance on the development of RV-specific B cells. Finally, computational models of RV-specific vaccine responses will be developed, and the experimental results will be applied to iteratively test and refine these models to generate a predictive model of RV immunity. These results will lead to greater understanding of the development of immunity to RV and other enteric viral infections and identify key targets for intervention to improve oral vaccine performance around the world.